The first three steps in de novo pyrimidine biosynthesis in higher eukaryotes are catalyzed by the multifunctional protein "CAD." The aspartate transcarbamylase activity of this protein is inhibited by N-phosphonacetyl (L) aspartate (PALA). Our previous studies demonstrated that hamster cells selected to resist high PALA concentrations have 100-200 times the amount of CAD protein and CAD mRNA as the PALA-sensitive cells. CAD gene amplification mediates the increases in CAD protein and mRNA in all cases studied thus far. We are pursuing the mechanism of CAD gene amplification by enumerating some of the parameters which affect the structure and stability of amplified units. Our initial goal has been to develop rapid and sensitive methods for localizing the position of the CAD gene on mammalian chromosomes. Using the autoradiographic procedures described below, we can now detect 5-10 CAD genes after a one-week exposure. We have found that most cells resistant to high PALA concentrations contain the amplified CAD genes on an extended arm of a single chromosome. The amplified region contains many G-bands. Occasionally, 5-10 CAD genes are located on the short arms of several other chromosomes. Each amplified unit is estimated to be about 10 to the 6th power base pairs long, far larger than the approximately 3 times 10 to the 4th power base pair CAD gene. Double minute chromosomes have never been observed in these cells. We have also used molecular cloning technology to isolate a CAD gene from PALA-resistant cells. We have introduced this cloned gene into Chinese hamster cells (Urd- A) deficient in CAD activity and have shown that the injected gene restores the ability of these cells to grow in the absence of added uridine. Our long-range goal in these experiments is to introduce the purified gene into different regions of the Urd- A genome and amplify the donated genes in different locations. We will then study the effect of neighboring sequences on the size, stability, and cytogenetic characteristics of units of CAD gene amplification.